Lassa fever virus (LFV) and several other arenaviruses cause severe hemorrhagic fever and weaponized forms of these viruses pose a threat as agents of bioterrorism. We have developed a reverse genetic system for arenaviruses. Using this system we iriolecularly characterized the Arenavirus genome promoter and identified the virus small RING finger Z protein as the driving force of budding. These advances allow us to propose developing novel antiviral strategies to combat pathogenic arenaviruses by inhibiting two essential steps of the Arenavirus life cycle: 1) RNA synthesis mediated by the Arenavirus polymerase, and 2) virus budding. For this we propose: 1) Screen aminoglycoside-based libraries to identify small molecules that bind and functionally disrupt the Arenavirus genome promoter, thus inhibiting virus multiplication. 2) Use biochemical and genetic assays to identify Z-interacting cellular proteins. The role in Arenavirus budding of these Z-interacting cellular proteins will be assessed using disruption of gene expression mediated by RNA interference. We will screen combinatorial chemical libraries to identify small molecules capable of disrupting Z-cellular protein interactions required for Arenavirus budding. Selected candidate molecules will be studied for their antiviral efficacy against arenaviruses in cell culture systems. Validated molecules will be considered for studies using animal models of Arenavirus infection.